Oligonucleotides are polymers built up by polycondensation of ribonucleoside (RNA) or deoxyribonucleoside (DNA) phosphates.
Oligonucleotides can be assembled by repetitive addition of nucleotide monomer using solid-phase methods. Since the introduction of solid-phase synthesis [R. B. Merrifield, J. Am. Chem. Soc. 85 (1963) 2149], the following requirements have been worked out: (1) The solid support must be insoluble and preferably unswellable in the solvent used. (2) Functional groups on the solid support must allow covalent binding of the first nucleoside in a reproducible manner. (3) The solid support must be chemically inert to all reagents used during synthesis and deprotection. The most commonly used supports are controlled pore glass beads (CPG), silica, or polystyrene beads.
Below the synthesis cycle of the commonly used phosporamidite method is described:
1. Deprotection of the 5'-hydroxyl group in order to generate the parent hydroxyl compounds. This is normally done by treatment of the support with di- or trichloroacetic acid in an organic solvent (for removal of protecting groups)
2. The support is washed in order to remove traces of acid.
3. The 5'-hydroxyl group is reacted with the 3'-phosphoramidite moiety of properly protected incoming nucleotide (A, C, G or T) in the presence of an activator (e.g. tetrazole) to form a 3'-5'-phosphite triester.
4. Excess reagents are removed by washing with an appropriate solvent.
5. Unreacted 5'hydroxyl groups are blocked as acetates (capping).
6. The capping reagent is removed by washing.
7. The phosphite triester is then oxidated to the corresponding phosphate triester. This is normally done by the action of aqueous iodine.
8. The oxidation reagents are removed by washing.
The process is repeated until the desired oligonucleotide sequence has been synthesized. After synthesis, all protecting groups are removed and the oligonucleotide is cleaved from the solid support.
In the synthesis, defective oligonucleotides are produced as a consequence of several effects, prominently premature termination of synthesis, followed by capping, which results in 5' truncated molecules, and depurination during the synthetic cycles that is followed by strand scission during deprotection. Recently, attention has also been directed at the appearance of shorter, internally deleted products--so called n-1 and n-2 fragments [Temsamani et al, (1995 ), Nucleic Acids Research 23 (11), 1841-1844]; [Fearon et al, (1995) Nucleic acids Res., 23 (14), 2754-2761].
The need for pure oligonucleotides is exemplified by the requirement for high quality products in antisense therapy [Gelfi et al, (1996), Antisense and Nucleic Acid Drug Development, 6, 47-53], in routine diagnostics applications, or for physicochemical and structural studies [Agback et al, (1994) Nucleic Acids Res, 22 (8), 1404-12]. Also in molecular cloning impure oligonucleotides frequently reduce efficiency and complicate interpretation of results [McClain et al, (1986) Nucleic Acids Res. 14 (16), 6770]; [Nassal, (1988) Gene, 66 (2), 279-94].
Preparative gel electrophoresis provides the best resolution for purification of oligonucleotides. The method is however laborious, often leading to considerable loss of material, and it is poorly suited for automation and scale-up.
Chromatographic separation can solve some of these problems, offering a potential for scale-up with minimal losses and using fully automatized instruments. These positive aspects are off-set by the rather poor resolving power of most chromatographic systems. As a partial solution to this problem chromatographic separation of oligonucleotides labeled with affinity tags has been used. The commonly used trityl-on oligonucleotide separation on reversed-phase columns, or capture of 5'-thiol labelled or biotinylated oligonucleotides on respective thiol-affinity [Bannwarth et al, (1990), Helv. Chim. Acta, 73, 1139-1147] or avidin columns [Olejnik et al, (1996), 24 (2), 361-366] offer the possibility to isolate fragments with intact 5'-ends. However, the 5' part of depurinated molecules notoriously contaminate oligonucleotides purified by this method.
A mild basic system has been proposed for partial deprotection and cleavage of apurinic-sites with the oligonucleotides still bound to the solid support. In this manner the 5' ends of depurinated molecules can be discarded before the oligonucleotides are released from the support, followed by isolation of molecules with intact 5' ends [Horn et al, (1988), Nucleic acids Res, 16 (24), 11559-71]. In practice, this strategy was accompanied by a substantial loss of products, due to inadvertent release of oligonucleotides during cleavage of depurinated sites.
In WO92/09615 there is described the use of an alkoxysilyl group as a linker of the oligonucleotide to the support.
This linker is inert during the synthetic cycles and it resists conditions that cleave apurinic sites. The linker is finally cleaved from the solid support with tetra butyl ammonium fluoride (TBAF) to obtain, after reversed-phase separation of DMTr-containing material, an oligonucleotide with both 3'- and 5'- ends intact. However, synthesis of this support was laborious and inconvenient. Due to low reactivity of the functional group of the linker the degree of substitution of the support becomes low which leads to insufficient nucleoside loadings of the support. Thus, this method is not suitable for preparation of support useful for large scale synthesis.